The objectives of this project are to (1) use molecular and classical approaches to investigate pathogen-arthropod interactions of vector- borne diseases of human importance in the United States, (2) examine basic biological questions concerning the behavior of ticks and fleas that influence their ability to transmit infectious agents, and (3) to develop rapid and definitive diagnostic assays to detect and identify bacterial pathogens in their arthropod vector. Several accomplishments were made concerning our third objective during the last year. First, we developed a monoclonal antibody (H9826) that bound to the flagellar protein of Borrelia hermsii, an agent of tick-borne relapsing fever, but not to flagellin of any of the other species tested, which included B. parkeri, B. turicatae, B. coriaceae, B. anserina, B. burgdorferi, and Leptospira interrogans. This antibody bound efficiently to B. hermsii in an indirect immunofluorescence assay and was used to rapidly detect and identify this spirochete in the peripheral blood of experimentally infected mice and in the central ganglia of Ornithodoros hermsi ticks. We have recently used this antibody to rapidly identify B. hermsii in the blood of human patients acutely ill with relapsing fever. Second, we adapted the polymerase chain reaction (PCR) for identifying rickettsial infections in ticks. Our assay amplified a 500 base pair DNA fragment from the gene encoding the rOmp B protein of Rickettsia rickettsii. The assay amplified fragments of the predicted size from all spotted fever group rickettsiae tested but not from any typhus group rickettsiae. We specifically amplified rickettsial DNA from the hemolymph, saliva, and triturated leg tissues from live, partially fed ticks. Samples of infected ticks preserved in 70% EtOH were also suitable for amplification by PCR. Also, we have successfully amplified Yersinia pestis DNA in experimentally infected fleas, Xenopsylla cheopis. This assay will provide a long overdue rapid method for detecting plague bacteria in mammalian hosts and vector tissues.